Haplotype analysis in Preimplantation Genetic Testing for Special Monogenic Diseases Cases based on Nanopore Sequencing

Abstract

Abstract Background Most monogenic diseases resulting from pathogenic variants lack effective treatments. Genetic counseling and preimplantation genetic testing for monogenic/ single-gene diseases (PGT-M) are significant and effective methods to block the inheritance of pathogenic variants. However, conventional PGT-M, based on next-generation sequencing (NGS), has limitations to analyze haplotype linkage when dealing with special pathogenic variants, particularly under conditions of de novo variant or incomplete pedigree information. Methods We recruited four couples who underwent PGT-M cycles in our center. They are patients or carriers of alpha-thalassemia, beta-thalassemia, polycystic kidney disease-1, and spinocerebellar ataxia-1, respectively, with different variants (missense variants, insertions and deletions (InDels), large fragment deletions, and short tandem repeats (STRs)). And most of them have no complete pedigree information. According to NGS method, affected single-sperm or embryo was used as proband. Because of its long-read length, nanopore sequencing was performed in PGT-M for haplotype analysis only using the couples’ blood sample. And it was compared with NGS method. Results We directly constructed haplotypes from the flanking single nucleotide polymorphisms (SNPs) linked with the pathogenic variants. The results of haplotype linkage analysis according to nanopore sequencing were consistent with that using NGS method. And embryos transferred were confirmed unaffected by prenatal diagnosis. Conclusion In this work, we developed a promising PGT-M method in haplotype linkage analysis based on nanopore sequencing, which is applicable for various kinds of target variants without the requirements for probands or additional family members. It overcoming the limitations of NGS method.